Defects in the human cholesterol transportation pathway can create two ultimately fatal recessive diseases (Wolman's disease and Cholesterol Ester Storage disease or CESD). The responsible enzyme, lysosomal acid lipase or LAL, is unable carry out its cholesterol ester hydrolysis function, resulting in a severe decline in liver function for Wolman's disease patients and a rapid increase in both cholesterol and triglyceride levels in blood serum with increased incidence of atherosclerotic disease in CESD patients' vessels by early adulthood. Yet, no recommended or universal diagnostic assay exists for either of these two diseases, resulting in many missed cases. By using a specific LAL substrate or an existing irreversible inhibitor of LAL, we will be able to specifically assay LAL enzymatic activit in dried blood spots. Likewise, the development of a more sensitive DBS assay for the more rare mucopolysaccharidoses (MPS) types IVA and VI will greatly benefit screening and diagnosing deficiencies due to the low baseline activity of these enzymes. The use of dried blood spots (DBS) is a low cost, relatively long-lived source of the enzymes found in leukocytes. Mass spectrometry will serve to quantify the amount of cleaved substrate from daughter ion fragmentation (or MS/MS scans), providing a high sensitivity for even small changes in enzyme function, which is critical for minimizing false positives in clinical settings. The development of an MS/MS dried blood spot assay for LAL deficiencies as well as improved MPS IVA and VI MS/MS DBS assays will provide newborn screening centers a means to efficiently identify potential cases that require immediate intervention and potentially life-long therapy that will protect the lives of these affected children.